1. Field of the Invention
The present invention relates to a system for determining the volume of a liquid delivery. More particularly, the present invention relates to systems and related methods to test or calibrate liquid delivery devices using a sample solution having multiple distinct detectable light absorbance spectral features using one or more dyes to cover a volume range, or a dilution range, of interest.
2. Description of the Prior Art
All currently manufactured calorimetric systems for testing or calibrating liquid delivery devices (e.g., the PCS® system and the MVS® system, both offered by Artel, Inc. of Westbrook, Me., assignee of the present invention and application, and the Pipette Volume Calibration Kit offered by VistaLab of Mt. Kisco, N.Y.) use a multiplicity of sample solutions for the purpose of testing a wide range of deliverable solution volumes. The sample solution is delivered by the device being tested into a diluent in a measurement vessel, and the solutions are mixed before measuring the absorbance of the resulting mixture. A concentrated sample solution is used to test a small liquid delivery volume, and a more dilute sample solution is used to test a large liquid delivery volume. As used herein, a “vessel” is any vial, cell, bottle, microtiter plate or other type of container for retaining a fluid therein, whether such vessel is sealed or not. Also, the vessel may be designed for collecting optical absorbance measurements by use of a horizontal beam spectrophotometer (such as a conventional UV-Vis spectrophotometer like the Cary 5000, Varian, Inc., Palo Alto, Calif.) or a vertical beam spectrophotometer (such as a microtiter plate reader like the ELx800, BioTek Instruments, Winooski, Vt.).
In simplified terms, the PCS® system includes one or more devices and one or more solutions used to calibrate a single-channel liquid dispensing device. On the other hand, the MVS® system includes one or more devices and one or more solutions used to calibrate a multi-channel liquid dispensing device. The difference between the two systems relates to the calculations performed subsequent to conducting absorbance measurements at one or more wavelengths corresponding to features present in the spectrum of the solution(s).
In the case of one commercial embodiment of the Artel PCS® system, for example, four different concentrations of a sample solution, each targeting the same spectral feature, are provided to cover the volume range from 2 microliters (μl) to 5000 μl. Two additional concentrations of the same sample solution extend the range down to 0.1 μl. The reason for using different concentrations is to maintain a measurable signal level (absorbance change resulting from the delivery of an aliquot of sample solution or diluent) within a defined signal range over the full range of liquid delivery volumes. That is to say, each concentration of sample solution is manufactured to produce a final measurable absorbance within a defined absorbance range for any delivered volume that is within the volume range for which the solution is designed. All concentrations of sample solution of the PCS® system are designed to produce measurable absorbance values over the same absorbance range. The disadvantage to using a multiplicity of solutions is that the user needs to stock them, rotate stock to make sure that the solutions are within their expiration date, and then choose which one or ones to use, and to dispense the correct solution(s) into one or more vessels from which the delivery device can aspirate the correct solution. There is ample opportunity for error, waste of unused solutions from the vessels, and waste of time and unnecessary distraction from the job of testing the delivery device. It is thus desirable to devise a method that would reduce the number of sample solutions required to test the entire volume range that can be delivered by a liquid delivery device.
In addition to volume delivery testing, colorimetric measurement systems using multiple concentrations of the same sample solution can be used to test dilution protocols, which are commonly employed in life science laboratories. Such dilution protocols are often employed in drug discovery testing where a compound of interest is serially diluted across a microtiter plate. For example, the user dispenses 200 μL of sample solution into the first column of a microtiter plate and then aspirates 100 μL from that first column and dispenses it into 100 μL of diluent in the second column, creating a 1:2 dilution ratio of the sample solution in the second column. Serially repeating this process across all columns in the plate results in a range of dilutions from a 1:1 ratio up to a 1:2048 ratio. An effective dilution protocol system is described in pending U.S. patent application Ser. No. 11/854,594, filed Sep. 13, 2007, having as assignee the assignee of the present application. The entire content of the referenced Ser. No. 11/854,594 pending application is incorporated herein by reference. If the MVS® system were used to test the dilutions made in this example, five sample solutions would be needed to cover the entire range of dilution ratios because each sample solution can only test a four- or five-fold range of dilution ratios before the absorbance signal of the single spectral feature in the sample solution is too low to be measured by the microtiter plate reader. Thus, to measure all dilution steps of this process, the serial dilution protocol has to be repeated for each of the five sample solutions such that a limited range of the dilution ratio series is tested with each sample solution. That is to say, each of the five sample solutions is serially diluted using the defined protocol. For each of the sample solutions, only a portion of the produced dilutions will be within a measurable absorbance range, but the combination of data from all sample solutions will provide measurements for every step of the entire process. As a result, many test vessels are filled with sample solution to cover the entire dilution range of interest but only a fraction are actually used. Thus, there is a waste of solution and of time.
Alternative methods for testing serial dilution protocols include fluorescence, which covers a far greater range (1,000-10,000 fold greater range) of dilution ratios before the signal is too low to measure. However, the fluorescence method introduces variability because of the instability of fluorescent dyes due to photo-bleaching, quenching, etc, and also lacks the traceability to international standards of an absorbance-based photometric approach. Therefore, it is also desirable to devise an absorbance-based photometric method that would measure an extended range of dilution ratios using one sample solution containing multiple spectral features, each capable of testing a unique four or five fold range of dilution ratios as an alternative to fluorimetry testing of dilution processing as noted herein.
Other systems have been described for enhancing calibration accuracy. See, for example, U.S. Pat. No. 5,298,978 for “Pipette Calibration System” of Curtis et al., incorporated herein by reference, U.S. Pat. No. 4,354,376 for “Kit for Calibrating Pipettes” of Greenfield, and U.S. Pat. Nos. 6,741,365 and 7,187,455 for “Photometric Calibration of Liquid Volumes” of Curtis, also incorporated herein by reference. These systems have limitations resolved by the use in the present invention of a dye, or a plurality of dyes, capable of producing multiple distinct detectable light absorbance spectral features in a smaller set of sample solutions, wherein each distinct detectable light absorbance spectral feature allows calibration or testing over a specific volume range. A dye is any molecule or chemical compound that imparts one or more features to the absorbance spectrum of the solution. This definition is not intended to limit the feature to the visible region of the spectrum, nor to limit the spectral feature to the dye or dyes alone. The solvent could also impart a spectral feature that could be used as is described in this invention. One skilled in the art may recognize that a dye may be the functional equivalent of a chromophore. As used herein, “distinct detectable light absorbance spectral features” are distinct and detectable peaks, valleys, plateaus, or any combination of peaks and valleys and plateaus in the absorbance spectrum of a solution under test. In the case of U.S. Pat. No. 4,354,376 of Greenfield, there is only one distinct detectable light absorbance spectral feature. In the case of U.S. Pat. No. 5,298,978 of Curtis et al., there is a second feature present in the spectrum of the diluent solely for the purpose of determining the pathlength of light through the vessel before any sample solution is added to the vessel, minimizing uncertainty in the results due to uncertainty in vessel dimensions. This second spectral feature is not contained in the sample solution dispensed with the liquid delivery device under test. In the case of U.S. Pat. Nos. 6,741,365 and 7,187,455 to Curtis, two spectral features are used to eliminate uncertainty in results that would otherwise occur due to uncertainty in the volume of diluent added to the tapered wells of the microtiter plate used as a measurement vessel. Both spectral features are contained in the sample solutions, however the function of the second spectral feature (in that particular case, CuCl2) is to correct for the unknown volume of diluent, not to cover a wider range of dispensing volumes.
What is needed is a system and related method to resolve the limitations of the existing liquid delivery measurement systems in which the use of colorimetric techniques to evaluate liquid delivery devices or protocols requires the use of an excess number of sample solutions for volume and dilution ranges of interest.